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2047 Enabling Potent T Cell Delivery and Mitigating Key Off-Target Concerns of In Vivo

CAR T Lentiviral Vectors with the Targeted Paramyxovirus Fusogen System

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Shirisha Amatya, MS1*, Jesse Green, PhD1*, Chi-Shuen Chu, PhD2*, Adam Hilterbrand, PhD2*, Reuben Burch1*, Chris Baldeviano, PhD3*, Edward Rebar, PhD4*, Terry J Fry, MD1, Albert Ruzo, PhD1*, Kutlu G Elpek, PhD5* and Kyle Trudeau, PhD5*

1Sana Biotechnology, Seattle, WA
2Sana Biotechnology, Seattle
3Sana Biotechnology, Cambridge
4Sana Biotechnology, Richmond, CA
5Sana Biotechnology, Cambridge, MA

The promise of in vivo CAR T is to achieve or exceed the efficacy of ex vivo CAR Ts with a systemically delivered vector, enabling off-the-shelf CAR T therapy without lymphodepletion. Ex vivo approaches generally incorporate cell enrichment or selection steps so that only T cells are modified and delivered to the patient. Accordingly the burden for in vivo approaches will be to limit off-target (non-T cell) delivery and mitigate any consequent risk of CAR delivery to non-T cells. Different approaches have been used to engineer vectors that can mediate cell-specific delivery, including lentiviral vectors (LVVs), but it remains unclear what features of the targeting approaches are critical for controlling specificity upon systemic delivery in vivo. At Sana we have leveraged targeted paramyxovirus fusogens to re-target LVVs to T cells. This fusogen system is distinct from endosomal escape-based systems in that cell entry is directly coupled to target receptor binding and is independent of pH-driven endosomal escape. Here we show how the entry mechanism of the paramyxovirus fusogen, compared to endosomal pH-dependent fusogens such as blind-VSVG, is critical to avoid key mechanisms of off-target delivery that can occur in vivo with alternative LVV-based delivery systems.

The first key mechanism of off-target delivery is non-specific vector uptake by phagocytic cells. The targeted paramyxovirus system enables potent and target-specific transduction of T cells with minimal delivery to a diverse panel of off-target phagocytic cells in vitro, including primary macrophages, as compared to substantial macrophage delivery with pH-dependent fusogens. The second off-target mechanism is B cell transduction that can lead to antigen masking and resistance to CAR T therapy. With LVVs, off-target B cell binding and delivery can be mediated by CD19-directed CAR protein passively incorporated onto the LVV during production. We show that CAR protein is incorporated onto LVVs with either fusogen system, which can mediate binding to B cells. We assessed CAR-mediated transduction potential in a permissive cell line engineered to express CD19. The paramyxovirus fusogen shows minimal (2-fold) increase in CAR-mediated delivery in this CD19-expressing line compared to the same cell line lacking CD19, while the pH-dependent fusogen shows a ~300-fold increase. Importantly, we show that detection of transduced CD19-expressing cells can be missed by flow cytometry due to masking of CAR detection in CD19-expressing cells. Our findings show that integrated vector copy number (VCN) assessment must be used. Overall the results show that paramyxovirus fusogens minimize CAR-mediated off-target transduction since entry is coupled to the targeted fusogen protein, whereas pH-dependent fusogens mediate entry via decoupled binders on the vector.

To confirm that the targeted paramyxovirus fusogen is specific to target CD8 T cells and does not transduce primary B cells, we assessed gene transfer in PBMC cell populations after exposure to vector. VCN assay was used to detect the presence of integrated LVV transgene in genomic DNA extracted from individually-sorted cell populations after 4-day culture in non-activating conditions. High VCN was detected in isolated CD8+ but not CD4+ T cells, and VCN was also not detected in isolated B cells from all 3 donors. Finally, to assess the on vs off-target delivery of the paramyxovirus fusogen system in an immunocompetent model, we performed a safety and biodistribution study in nemestrina NHPs with our CD8-targeted LVV delivering CD19 CAR transgene, called SG299. NHPs were dosed i.v. with 1 x 109 or 4 x 109 IU/kg. High VCN signal was detected through day 34 in all animals dosed at 4 x 109 IU/kg in CD8-enriched PBMCs (0.0032 - 0.6544 vc/dg, vector copy per diploid genome). Tissues analyzed at day 90 showed quantifiable VCN in target tissues including spleen and lymph node. Importantly, off-target tissues including liver, testis, kidneys, and heart, among others, showed undetectable VCN.

In summary, we show that the unique cell entry mechanism of the targeted paramyxovirus fusogen system mitigates key off-target concerns with in vivo delivery of LVVs. Accordingly our in vivo CAR T vector, SG299, shows highly specific and potent delivery to CD8 T cells in NHPs. Thus SG299 represents a differentiated approach for enabling in vivo CAR T therapy, potentially avoiding key off-target concerns.

Disclosures: Amatya: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Green: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Chu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Hilterbrand: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Burch: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Baldeviano: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Rebar: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: United States Patent and Trademark Office: Patents & Royalties: WO2019178382A1; United States Patent and Trademark Office: Patents & Royalties: WO2015084513A1; Sana Biotechnology: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months; United States Patent and Trademark Office: Patents & Royalties: WO2017205747A1. Ruzo: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Elpek: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Trudeau: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

*signifies non-member of ASH